Abstract The rate of reaction between superoxide anion (O ¯. 2) and 1,2-dihydroxybenzene-3,5-disulfonic acid (tiron) was measured with pulse radiolysis-generated O ¯. 2. A kinetic spectrophotometric method utilizing competition between p-benzoquinoneand tiron for O ¯. 2 was employed. In this system, the known rate of reduction of p-benzoquinonewas compared with the rate of oxidation of tiron to the semiquinone. From the concentration dependence of the rate of tiron oxidation, the absolute second order rate constant for the reaction was determined to be 5 · 10 8 M −·s −1. Ascorbat reduced O ¯. 2 to hydrogen peroxide with a rate constant of 10 8 M −1 · s −1 as determined by the same method. The tiron semiquinone may be used as an indicator free radical for the formation of superoxide anion in biological systems because of the rapid rate of oxidation of the catechol by O ¯. 2 compared to the rate of O ¯. 2 formation in most enzymatic systems. Tiron oxidation was used to follow the formation of superoxide anion in swollen chloroplasts. The chloroplasts photochemically reduced molecular oxygen which was further reduced to hydrogen peroxide by tiron. Tiron oxidation specifically required O ¯. 2 since O 2 was consumed in the reaction and tiron did not reduce the P 700 cation radical or other components of Photosystem I under anaerobic conditions.